Cleavage of Adenovirus Type 2 DNA into Six Unique Fragments by Endonuclease R·RI

The DNA of adenovirus type 2 was cleaved by restriction endonuclease R.RI into six fragments. These fragments were separated by electrophoresis on composite agarose-polyacrylamide gels. Their molecular weights ranged from 1.1 x 10(6) to 13.6 x 10(6), as measured by electron microscopy. Each fragment represented a unique segment of adenovirus type 2 DNA since: (i) the fragments were obtained in equimolar amounts; (ii) the sum of their molecular weights was equal to the molecular weight of complete adenovirus DNA; and (iii) each fragment exhibited a rate of renaturation that was inversely proportional to its size.     

Alteration of the Relative Stability of dA·dT and dG·dC Base Pairs in DNA

Several small alkylammonium ions can eliminate, or even reverse, the usual dependence of the DNA transition temperature on base composition. For example, in 3 M tetramethylammonium chloride, or 2.4 M tetraethylammonium chloride, DNAs of different base compositions all melt at a common temperature, and with a greatly decreased breadth of transition reflecting only the sequence-independent components of melting cooperativity. At still higher concentrations of such additives, dG.dC-rich DNAs melt at lower temperatures than dA.dT-rich molecules. Circular dichroism spectra show that these additives alter the structure of the DNA double helix very little at room temperature. This differential (base-specific) effect on helix stability is investigated with several small additives related to the tetraalkylammonium ions. Additives larger than tetraethylammonium ion have little differential effect on helix stability. Preferential binding of ions to dA.dT base pairs, requiring fit into a "groove" of DNA, is consistent with these data and with equilibrium binding studies. These differential effects can be distinguished from general destabilizing effects, which are independent of specific features of macromolecular conformation or chemistry. Possible experimental uses of this ability to alter the base-composition-dependent components of the stability of the DNA helix are discussed, as well as the insight this phenomenon provides into the molecular basis for the differential stability of dA.dT and dG.dC base pairs.     

Preparation and Characteristics of Na2HPO4·12H2O-K2HPO4·3H2O/SiO2 Composite Phase Change Materials for Thermal Energy Storage

In this paper, a series of eutectic hydrated salts was obtained by mixing Na₂HPO₄·12H₂O (DHPD) with K₂HPO₄·3H₂O (DHPT) in different proportions. With the increase in the content of DHPT, the phase transition temperature and melting enthalpy of eutectic hydrated salts decreased gradually. Moreover, the addition of appropriate deionized water improved the thermal properties of eutectic hydrated salts. Colloidal silicon dioxide (SiO₂) was selected as the support carrier to adsorb eutectic hydrated salts, and the maximum content of eutectic hydrated salts in composite PCMs was 70%. When the content of the nucleating agent (Na₂SiO₃·9H₂O) was 5%, the supercooling degree of composite PCMs was reduced to the minimum of 1.2 °C. The SEM and FT-IR test results showed that SiO₂ and eutectic hydrated salts were successfully combined, and no new substances were formed. When the content of DHPT was 3%, the phase transition temperature and melting enthalpy of composite PCMs were 26.5 °C and 145.3 J/g, respectively. The results of thermogravimetric analysis and heating–cooling cycling test proved that composite PCMs had good thermal reliability and stability. The application performance of composite PCMs in prefabricated temporary houses was investigated numerically. The results indicated that PCM panels greatly increased the Grade I thermal comfort hours and reduced energy consumption. Overall, the composite PCM has great development potential building energy conservation.     

A Simple and Efficient Method for Preparing High-Purity α-CaSO4·0.5H2O Whiskers with Phosphogypsum

A simple and efficient approach for the high-purity CaSO₄·2H₂O (DH) whiskers and α-CaSO₄·0.5H₂O (α-HH) whiskers derived from such phosphogypsum (PG) was proposed. The impact of different experimental parameters on supersaturated dissolution–recrystallization and preparation processes of α-CaSO₄·0.5H₂O was elaborated. At 3.5 mol/L HCl concentration, the dissolution temperature and time were 90 °C and 20 min, respectively. After eight cycles and 5–8 times cycles, total crystallization amount of CaSO₄·2H₂O was 21.75 and 9.97 g/100 mL, respectively, from supersaturated HCl solution. The number of cycles affected the shape and amount of the crystal. Higher HCl concentration facilitated CaSO₄·2H₂O dissolution and created a much higher supersaturation, which acted as a larger driving force for phase transformation of CaSO₄·2H₂O to α-CaSO₄·0.5H₂O. The HCl solution system’s optimum experimental conditions for HH whiskers preparation involved acid leaching of CaSO₄·2H₂O sample, with HCl concentration 6.0 mol/L, reaction temperature 80 °C, and reaction time 30 min–60 min. Under the third cycle conditions, α-CaSO₄·0.5H₂O whiskers were uniform in size, clear, and distinct in edges and angles. The length range of α-CaSO₄·0.5H₂O whiskers was from 106 μm to 231 μm and diameter range from 0.43 μm to 1.35 μm, while the longest diameter ratio was 231. Purity of α-CaSO₄·0.5H₂O whiskers was approximately 100%, where whiteness reached 98.6%. The reuse of the solution enables the process to discharge no waste liquid. It provides a new reference direction for green production technology of phosphogypsum.     

Increased Susceptibility of Cells Treated with Interferon to the Toxicity of Polyriboinosinic·Polyribocytidylic Acid

Concentrations of the synthetic polymer polyriboinosinic.polyribocytidylic acid that produced no detectable toxicity in normal L cells produced marked cytotoxicity in L cells treated with interferon. This increase in the susceptibility of cells to the toxicity of the polymer was also observed in human cells and secondary mouse embryo cells treated with homologous interferons before exposure to the polynucleotides. The degree of enhancement of toxicity was dependent on the concentration of interferon to which the cells were exposed. The ratio of antiviral activity induced by interferon to enhancement of toxicity by interferon remained constant through about 1000-fold purification. Various interferon preparations induced by viruses or by polyriboinosinic.polyribocytidylic acid in vivo or in vitro, and international reference standard interferons all exhibited enhancement of toxicity. Both enhancement of toxicity and antiviral activity were destroyed by trypsin and by incubation at 56 degrees for 1 hr, did not act on heterologous cells, were not sedimented by ultracentrifugation, and were not inactivated by ribonuclease, deoxyribonuclease, irradiation with ultraviolet light, or exposure to a pH of 2.     

Evidence of sp2-like Hybridization of Silicon Valence Orbitals in Thin and Thick Si Grown on α-Phase Si(111)√3 × √3R30°-Bi

One-monolayer (ML) (thin) and 5-ML (thick) Si films were grown on the α-phase Si(111)√3 × √3R30°-Bi at a low substrate temperature of 200 °C. Si films have been studied in situ by reflection electron energy loss spectroscopy (REELS) and Auger electron spectroscopy, as a function of the electron beam incidence angle α and low-energy electron diffraction (LEED), as well as ex situ by grazing incidence X-ray diffraction (GIXRD). Scanning tunneling microscopy (STM), and scanning tunneling spectroscopy (STS) were also reported. The REELS spectra, taken at the Si K absorption edge (~1.840 KeV), reveal the presence of two distinct loss structures attributed to transitions 1s→π* and 1s→σ* according to their intensity dependence on α, attesting to the sp²-like hybridization of the silicon valence orbitals in both thin and thick Si films. The synthesis of a silicon allotrope on the α-phase of Si(111)√3 × √3R30°-Bi substrate was demonstrated by LEED patterns and GIXRD that discloses the presence of a Si stack of 3.099 (3) Å and a √3 × √3 unit cell of 6.474 Å, typically seen for multilayer silicene. STM and STS measurements corroborated the findings. These measurements provided a platform for the new √3 × √3R30° Si allotrope on a Si(111)√3 × √3 R30°-Bi template, paving the way for realizing topological insulator heterostructures from different two-dimensional materials, Bi and Si.     

Analysis of mRNA populations by cDNA·mRNA hybrid-mediated inhibition of cell-free protein synthesis

Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA in vitro. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C(0)t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.     

Immunocytochemical Localization and Serologic Detection of Transforming Growth Factor β1

Objective. To determine the presence of transforming growth factor β1 (TGFβ1) and inflammatory cell markers (HLA–DR and Factor XIIIa) and to compare these with the presence of type I procollagen, in clinically uninvolved and involved skin from patients with different subsets of systemic sclerosis (SSc), and to analyze circulating levels of TGFβ1 in SSc patients. Methods. TGFβ1, HLA-DR, Factor XIIIa, and type I procollagen were detected in skin biopsy sections using a biotin–streptavidin–peroxidase system. Levels of circulating TGFβ1 were measured using a capture enzyme-linked immunosorbent assay technique. Results. Patients with active diffuse cutaneous SSc (dcSSc) showed minimal TGFβ1 but significant type I procollagen staining in involved skin, while the clinically uninvolved skin of these patients showed moderate extracellular and intra-epidermal TGFβ1 immunoreactivity. Patients with limited cutaneous SSc (IcSSc) showed elevated TGFβ1 staining in both involved and uninvolved skin, as well as procollagen staining. Significant TGFβ1 reactivity, HLA–DR and Factor XIIIa immunoreactivity, numerous inflammatory cells, and procollagen staining were seen in specimens from patients with morphea. Sequential biopsies suggested the presence of cytokine activity at the earliest stages of disease, which was not maintained with progression of sclerosis. Among the disease groups studied, elevated levels of circulating TGFβ1 were seen only in patients with morphea. Conclusion. The pattern of TGFβ1 staining in dermal sections from patients with dcSSc, IcSSc, and morphea suggests that this cytokine is important in the pathogenesis of scleroderma. Furthermore, the presence of TGFβ1 prior to the onset of fibrosis indicates an early involvement of this growth factor, possibly in the inflammatory stage of the disease.     

Obtention and Characterization of Ferrous Chloride FeCl2·4H2O from Water Pickling Liquors

As a hazardous waste, water pickling liquors must be properly treated. An alternative consists of promoting the formation of ferrous salts from this residue due to their higher ferrous content. Since FeCl₂·4H₂O is widely used in several applications, obtaining pure crystals of this material appears to be an interesting prospect. However, this compound has scarcely been investigated. In the present work, FeCl₂·4H₂O crystals were obtained from water pickling liquors. Their structural and morphological characteristics were investigated by X-ray diffraction, scanning electron microscopy as well as Mössbauer spectroscopy. In addition, the photoluminescence study of the obtained samples was also assessed. It was observed that after some aging time, the obtained crystals changed in colour from green to more yellowish. As such, the aged sample was also evaluated, and their structural characteristics were compared with the original crystals. Despite this, the obtained crystals exhibit a FeCl₂·4H₂O structure, which is not modified with the aging of the sample.     

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Chimeric antigen receptor (CAR)–engineered T cells have invigorated the field of cancer immunotherapy with their proven ability to treat CD19⁺ malignancies in the clinic (1–4) and continuing progress in solid tumors (5, 6). The synthetic CAR imparts T cells with the ability to recognize antigen independent of peptide presentation by major histocompatibility complexes. This antigen recognition is most often mediated by single-chain variable fragments derived from monoclonal antibodies. As an alternative, naturally occurring ligands or receptors have been used for CAR antigen recognition (7), including interleukin 13 (IL13) (8–10), a proliferation-inducing ligand (APRIL) (11), NKG2D (12), NKp44 (13), and CD27 (14). By leveraging natural binding interactions, these molecules can mediate CAR antigen recognition with minimal additional engineering (8, 14), are fully human in sequence and thus carry potentially lower immunogenicity than other classes of engineered antigen binding domains, and can potentially target multiple cancer biomarkers (11–13). However, the ability to target multiple receptors can also be disadvantageous when binding partners are not implicated in disease.IL13 is one prominent example of a naturally occurring ligand that has been used for CAR antigen recognition (8–10). IL13 interacts strongly with the high-affinity receptor IL13Rα2 (15), which is a versatile therapeutic target due to its rare expression in normal tissue (16) and overexpression in many human cancers, including glioblastoma (GBM) (17), pancreatic ductal adenocarcinoma (18), melanoma (19), ovarian carcinoma (20), clear cell renal cell carcinoma (21), breast cancer (22), and lung cancer (23). A second IL13 receptor family member, IL13Rα1, interacts with IL13 with lower affinity (15) and is ubiquitously expressed in healthy tissue (16). Additionally, IL4Rα can stabilize the IL13Rα1-IL13 complex (15) to mediate signaling through the JAK/STAT6 pathway (24). This receptor pair is coexpressed in pulmonary and other normal tissues (25). Despite this wide expression of IL13 binding partners in healthy tissue, an IL13 ligand–based CAR has shown safety in humans during clinical trials with locoregional central nervous system delivery in GBM (5, 26), suggesting that toxicity from on-target/off-disease binding is not problematic in this context. However, for the treatment of systemic disease, the wide expression of IL13 binding partners outside of the diseased tissue could act as a sink for IL13-based therapy, resulting in safety concerns and possibly impeding trafficking to the disease site. Previous work in the field has attempted to address this problem by generating CARs derived from IL13 variants containing mutations to direct binding away from IL13Rα1/IL4Rα. Mutations at E12 have yielded improved selectivity for IL13Rα2 over IL13Rα1 (8, 9), albeit with the E12Y mutation still allowing measurable recognition of IL13Rα1 in the context of both recombinant antigen and antigen-expressing cancer cells (9). The addition of both E12K and R109K mutations into an IL13-based CAR also showed attenuated, but not abolished, recognition of IL13Rα1-expressing cancer cells relative to IL13Rα2-expressing cancer cells (10). While these examples are encouraging, additional protein engineering is warranted to develop an IL13Rα2-specific IL13 mutant.Structure-based protein engineering and directed evolution approaches offer opportunities to modify the affinity and specificity of binding interactions (27, 28). In this approach, structural information is used to identify residues that contribute to binding interactions, combinatorial libraries are developed through designed or random mutation at the identified residues, and high-throughput in vitro methods are employed to screen for the desired function. Previous applications of this method in the context of cytokines have led to the development of a panel of IL13 mutants with a 6-log affinity range for IL13Rα1 to study the interplay of binding affinity and signal transduction (29), engineering of an orthogonal interleukin 2 (IL2) cytokine-receptor complex system that does not act with the native cytokine or receptor (30), and the development of transforming growth factor beta (TGFβ)-based inhibitors (31), among other examples.Here, we describe the development of IL13-mutein CARs with improved selectivity for IL13Rα2 relative to IL13Rα1 and study their activity in IL13Rα1-expressing, IL13Rα2-expressing, and IL13Rα1/IL4Rα-coexpressing contexts. Prior knowledge of the structures of the IL13 complexes with IL13Rα2 and IL13Rα1/IL4Rα (15) informed the design of an IL13-mutein library that was screened using yeast surface display for diminished binding to IL13Rα1 (29). Characterization of hits yielded two promising candidates, termed C4 and D7, with markedly improved selectivity for IL13Rα2, as shown by affinity characterization. These IL13 muteins were then built into CAR constructs for functional comparison to CARs derived from IL13 wild-type (WT) and IL13 with the E12Y mutation. In vitro and in vivo functional characterization of C4 and D7 IL13-mutein CAR T cells showed decreased activation, degranulation, cytokine release, and cytolytic activity compared to WT and E12Y CAR T cells in the presence of IL13Rα1-expressing cancer cells. Interestingly, C4, but not D7, showed attenuated cytotoxicity relative to WT against IL13Rα1/IL4Rα-coexpressing cancer cells in vitro and in vivo. Conversely, all of the IL13-mutein CAR T cells exhibited similar cytolytic killing of IL13Rα2 targets in vitro and in vivo. Collectively, this work provides insight into the interplay of binding affinity and selectivity in CAR T cell activity and validates IL13-mutein CARs with improved recognition profiles for targeting IL13Rα2-expressing malignancies. Application of these CARs could expand the therapeutic window for systemic administration of IL13Rα2-targeted therapy for a variety of cancers.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.     

Effects of α tocopherol and β carotene supplements on symptoms, progression, and prognosis of angina pectoris

Objective—To evaluate the effects of α tocopherol and β carotene supplements on recurrence and progression of angina symptoms, and incidence of major coronary events in men with angina pectoris.
Design—Placebo controlled clinical trial.
Setting—The Finnish α tocopherol β carotene cancer prevention study primarily undertaken to examine the effects of α tocopherol and β carotene on cancer.
Subjects—Male smokers aged 50-69 years who had angina pectoris in the Rose chest pain questionnaire at baseline (n = 1795).
Interventions—α tocopherol (vitamin E) 50 mg/day, β carotene 20 mg/day or both, or placebo in 2 × 2 factorial design.
Main outcome measures—Recurrence of angina pectoris at annual follow up visits when the questionnaire was readministered; progression from mild to severe angina; incidence of major coronary events (non-fatal myocardial infarction and fatal coronary heart disease).
Results—There were 2513 recurrences of angina pectoris during follow up (median 4 years). Compared to placebo, the odds ratios for recurrence in the active treatment groups were: α tocopherol only 1.06 (95% confidence interval (CI) 0.85 to 1.33), α tocopherol and β carotene 1.02 (0.82 to 1.27), β carotene only 1.06 (0.84 to 1.33). There were no significant differences in progression to severe angina among the groups given supplements or placebo. Altogether 314 major coronary events were observed during follow up (median 5.5 years) and the risk for them did not differ significantly among the groups given supplements or placebo.
Conclusions—There was no evidence of beneficial effects for α tocopherol or β carotene supplements in male smokers with angina pectoris, indicating no basis for therapeutic or preventive use of these agents in such patients.

Keywords: antioxidants;  angina pectoris;  prevention;  vitamin supplements     

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson’s disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder caused by loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase). GCase degrades glycosphingolipids (GSLs), including glucosylceramides (GluCers), into glucose and ceramide, and GCase mutations result in the accumulation of GluCer in lysosomes of various tissues. Heterozygote carriers of the same loss-of-function GCase mutations are estimated to be at 5- to 10-fold higher risk for developing Parkinson’s disease (PD) or Lewy body dementia (1). In GD, significant variability exists in the clinical and pathological presentation, resulting in three main GD subtypes (2). Type 1 GD is characterized by visceral abnormalities, including enlarged liver and spleen and bone marrow dysfunction, leading to thrombocytopenia but without neurodegeneration and α-synuclein (α-syn) pathology (3). Types 2 and 3 demonstrate similar visceral symptoms but with additional extensive neuronal loss, α-syn pathology in the form of classical Lewy bodies, and neurological dysfunction (3, 4). As life expectancy of type 1 GD has increased because of enzyme replacement therapy, a higher percentage of patients develop PD symptoms with age (5), suggesting that aging could contribute to the penetrance of GBA1 mutations. The dramatic phenotypic heterogeneity suggests that GD is not a simple, monogenic disease but a complex disorder that is influenced by both genetic and nongenetic modifiers. Although the factors that contribute to clinical and pathological variability in GD are not known, genetic modifiers have been identified that associate with GD severity, including CLN8 and SCARB2 (6, 7). Within PD patients that harbor GBA1 mutations (GBA-PD), the search for genetic modifiers has shown that synergism may exist with the SNCA gene that encodes α-syn and CTSB that encodes lysosomal cathepsin B (8). Variants in lysosomal cathepsins could influence the severity of α-syn accumulation, since, under physiological or pathological conditions, α-syn can be degraded by the lysosome (9–11) and is a direct substrate of cathepsin B and L (12).An additional factor that may contribute to phenotypic variability in GD is the accumulation of specific GluCer subtypes with particular acyl chain lengths. GluCer and other GSLs exist as a family of lipid isoforms differentiated by the length of the N-acyl fatty acid moiety linked to the sphingoid base. GluCer chains range from C14 to C26 in the brain; however, C18 and C24:1 are the predominant species (13). Studies of neuronopathic GD (nGD) brain or mouse models showed intraneuronal accumulation of multiple GluCer species that correlated with neuroinflammation (14–19), and some cases demonstrate selective accumulation of long-chain GluCers in nGD (20). Our recent work in PD patient midbrain neurons showed that inhibition of wild-type (wt) GCase, caused by α-syn, resulted in the selective accumulation of long-chain-length GluCers, including C22 and C24:1, while C14, 16, and C18 were unchanged (21). Together, these data indicate that GluCer accumulation plays an important role in neurodegeneration induced by GBA1 mutations; however, the specific contributions of distinct GluCer species have not been examined.Here, we extend our studies on the role of GSLs in α-syn aggregation to further define conditions that are required to induce pathology and neurological dysfunction. We previously showed that α-syn exists as monomers and high–molecular weight (HMW) oligomers under physiological conditions in human midbrain cultures (22). In vitro, we found that GluCer mildly induced aggregation of α-syn monomers but primarily acted on physiological oligomers to convert them into toxic oligomers and fibrillar inclusions (22). α-syn accumulation can be prevented or reversed by reducing GSLs with GluCer synthase inhibitors (GCSi) in both GD and PD patient cultures, as well as in mouse models (22–24). While this work suggests a close relationship between GCase function and α-syn pathology, additional factors must exist that create a permissive environment for α-syn accumulation. Indeed, studies that used newborn mice or embryonic primary neuron cultures treated with the GCase inhibitor, conduritol beta epoxide (CBE), have shown no changes in α-syn despite reduced GCase activity (25–27). However, other studies that use matured neuron cultures, neuronal cell lines, or adult mice have shown that CBE dramatically induces α-syn aggregates (22, 28–31). We used an in vivo GD model and induced pluripotent stem cell (iPSC)–derived patient midbrain cultures to identify specific conditions that are required to induce α-syn pathology, providing possible explanations for the variable neurological penetrance in patients that harbor GBA1 mutations.     

Serum concentrations of α tocopherol, β carotene, and retinol preceding the diagnosis of rheumatoid arthritis and systemic lupus erythematosus

OBJECTIVES—Because oxidative damage has been implicated in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus, this study was designed to see if serum concentrations of α tocopherol, β carotene, and retinol, substances believed to be involved in the prevention or repair of oxidative damage, might be lower among persons who develop rheumatoid arthritis or systemic lupus erythematosus than among those who do not.
METHODS—For this prospective case-control study, persons with rheumatoid arthritis and systemic lupus erythematosus that developed two to 15 years after donating blood for a serum bank in 1974 were designated as cases. For each case, four controls were selected from the serum bank donors, matched for race, sex, and age. Stored serum samples from cases and controls were assayed for α tocopherol, β carotene, and retinol.
RESULTS—Cases of both diseases had lower serum concentrations of α tocopherol, β carotene, and retinol in 1974 than their matched controls. For rheumatoid arthritis, the difference for β carotene (−29%) was statistically significant.
CONCLUSIONS—These findings support those of a previous study that low antioxidant status is a risk factor for rheumatoid arthritis. They suggest a similar association for systemic lupus erythematosus.